Rabbit anti et1

Protein was drawn from 20 mg lung tissue on ice by treatment with lysis buffer and phenylmethanesulfonyl fluoride (both from Beyotime, Shanghai, PRC). Protein supernatants were centrifuged at 10,000 rpm for 10 minutes (4°C) to remove tissue fragments of the sediments. Protein concentrations were determined using a Bio-Rad protein-assay instrument, and the protein was boiled with loading buffer. Equal amounts of protein from all the lung tissues were dissolved in sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis (PAGE) sample buffer, separated in SDS-PAGE, and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with the respective primary antibodies (rabbit anti-HMGB1 and rabbit anti-ET1; both from Abcam PLC, Cambridge, UK) overnight at 4°C after being blocked with 5% fat-free milk for 2 hours. The blots were incubated with secondary antibodies conjugated to horseradish peroxidase for 1 hour at room temperature with continuous shaking. After washing, the protein blots were detected using an enhanced chemiluminescence kit (EMD Millipore, Billerica, MA, USA) and exposed to X-ray film. Bands were quantified using FluorChem 9900 (ProteinSimple, San Jose, CA, USA), and β-actin was used as an internal reference.